This research used "operation-and-interview" to analyze the microscope operative skills and the conceptional cognition of the elementary and junior high school students, and based on the theory of the two-tier multiple-choice instrument of the world under microscope (TIWM). The purpose of the instrument is to testify the rationale of students' answers. The content revealed that TIWM was divided into two parts:( 1 )elementary school test papers which included the microscope operative skills, recognizable characteristics of living things, microorganism with living environment, and micro-object in environment;(2)junior high school test papers which included all parts, except micro-object in environment. This research also took grade 7 and grade 5 students as subjects in Taipei areas, simultaneously. The result revealed that the reliability of internal quality for TIWM was 0.98( elementary school test papers )and 0.85 (junior high school test papers ); the retested reliability for TIWM was 0.81 ( elementary school test papers)and 0.67(junior high school test papers ). This research also built up validity of content, validity of profession and two-tier factorial analysis of TIWM The research found that reliability and validity of the test papers of two-tier multiple-choice instrument of "the world under microscope" (TIWM) were good on the elementary school, a little low on the junior school, and adequate on the rest.